title: Recruitment of trimeric proliferating cell nuclear antigen by G1-phase cyclin-dependent kinases following DNA damage with platinum-based antitumour agents.
abstract: BACKGROUND
  In cycling tumour cells, the binary cyclin-dependent kinase Cdk4/cyclin D or Cdk2/cyclin E complex is inhibited by p21 following DNA damage to induce G1 cell-cycle arrest. However, it is not known whether other proteins are also recruited within Cdk complexes, or their role, and this was investigated.
  
  
  METHODS
  Ovarian A2780 tumour cells were exposed to the platinum-based antitumour agent 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP), which preferentially induces G1 arrest in a p21-dependent manner. The Cdk complexes were analysed by gel filtration chromatography, immunoblot and mass spectrometry.
  
  
  RESULTS
  The active forms of Cdk4 and Cdk2 complexes in control tumour cells have a molecular size of ~140 kDa, which increased to ~290 kDa when inhibited following G1 checkpoint activation by DAP. Proteomic analysis identified Cdk, cyclin, p21 and proliferating cell nuclear antigen (PCNA) in the inhibited complex, and biochemical studies provided unequivocal evidence that the increase in ~150 kDa of the inhibited complex is consistent with p21-dependent recruitment of PCNA as a trimer, likely bound to three molecules of p21. Although p21 alone was sufficient to inhibit the Cdk complex, PCNA was critical for stabilising p21.
  
  
  CONCLUSIONS
  G1 Cdk complexes inhibited by p21 also recruit PCNA, which inhibits degradation and, thereby, prolongs activity of p21 within the complex.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Unit 1950, 1515 Holcombe Boulevard, Houston, TX, USA.
authors.forename: G
authors.initials: G
authors.lastname: He
authors.forename: J
authors.initials: J
authors.lastname: Kuang
authors.forename: J
authors.initials: J
authors.lastname: Koomen
authors.forename: R
authors.initials: R
authors.lastname: Kobayashi
authors.forename: A R
authors.initials: AR
authors.lastname: Khokhar
authors.forename: Z H
authors.initials: ZH
authors.lastname: Siddik
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D056741:Cyclin D / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D016193:G1 Phase / Q000187:drug effects* / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D061986:MCF-7 Cells; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D014407:Tumor Cells, Cultured
chemical_list: C119451:(diaminocyclohexane)(diacetato)(dichloro)platinum; D000970:Antineoplastic Agents; D056741:Cyclin D; D019927:Cyclin E; D009944:Organoplatinum Compounds; D018809:Proliferating Cell Nuclear Antigen; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4